Takiko Shimada

Does Decrease in Ribulose-1,5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO2 and Light in Rice Plants?

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100–115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.

Physicochemical properties of amylose-free starch from transgenic sweet potato

A transgenic amylose-free sweet potato has been obtained by introduction of granule-bound starch synthase I (GBSSI) cDNA of sweet potato in sense orientation (Plant Cell Reports (2001)). In this study, starches from 6 transgenic sweet potatoes produced by introduction of GBSSI cDNA, including the amylose-free transformant, were analyzed for their physicochemical properties, granule-size distribution, enzymatic digestibility, amylopectin structure, gelatinization properties and pasting properties. We observed little difference in granule size distribution between amylose-free starch and control starch. Amylose-free starch was more susceptible to glucoamylase digestion than control starch. The amylopectin of the amylose-free transformant was found to have a slightly lower content of short chains. Amylose-free starch showed higher gelatinization temperature and gelatinization enthalpy and lower setback in comparison with control starch. Thus, it was found that the starch from the amylose-free transgenic sweet potato possessed unique physicochemical properties.

Transformation of sweet potato (Ipomoea batatas (L.) Lam.) plants by Agrobacterium rhizogenes

Abstract Transgenic sweet potato plants were obtained after Agrobacterium rhizogenes -mediated transformation. Leaf disks of in vitro plants were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on leaf disks by both agropine-type and mikimopine-type strains. Whole plants transformed with Ri-T-DNA were regenerated from the hairy roots in five cultivars. These plants had wrinkled leaves, altered shape of flowers, reduced apical dominance, shortened internodes, small storage roots and abundant, frequently branching roots that showed reduced geotropism. Transgenic sweet potato plants possessing both NPT II gene and GUS gene were also obtained from the hairy roots by infection with Agrobacterium rhizogenes containing the binary vector pBI121 in addition to the wild-type Ri-plasmid.

Whole-plant growth and N allocation in transgenic rice plants with decreased content of ribulose-1,5-bisphosphate carboxylase under different CO2 partial pressures

Growth of transgenic rice (Oryza sativa L.) with an antisense gene to the small subunit of Rubisco was analysed under 36 and 100 Pa CO2 during a 14-h photoperiod (1000 mol quanta m–2 s–1). Two lines of the antisense plants were used; one with 65% wild-type Rubisco and the other with 40% wild-type Rubisco. The plants were grown hydroponically for 70 d. The final biomass of the antisense plants grown in 36 Pa CO2 was much smaller than that of the wild-type plant. However, several compen-sation phenomena were found in the antisense plants. Increased biomass allocation to leaf blades and preferential N investment in leaf blades were observed. Leaf senescence was also delayed. Elevated CO2 levels up to 100 Pa caused the antisense plants to achieve a size similar to that of the wild-type plant. However, although the antisense plant with 65% wild-type Rubisco was selected as a plant with optimal Rubisco content for CO2 -saturated photosynthesis, its final biomass was not greater than that of the wild-type plant. This may have been caused by a relatively strong Rubisco-antisense effect during the early stage of growth (21–42 d). N-use efficiency for growth after d 42 was greater in the selected antisense plant. Thus, improvement of N-use efficiency at the level of a single leaf did not necessarily lead to greater production of biomass at the whole-plant level.

Production of transgenic wheat through particle bombardment of scutellar tissues: Frequency is influenced by culture duration

Summary A transformation system for hexaploid wheat was developed by using scutellar tissues of cultured immature embryos via particle bombardment. The scutellar tissues of immature embryos were cultured for 1 to 9 d prior to bombardment with two plasmids, pBI221 or p Act1 -F, containing the s-glucuronidase (GUS) gene and pARK22 containing the bialaphos resistant gene ( bar ). Transient expression of the reporter GUS gene was not influenced by culture duration prior to transformation. On the other hand, transgenic wheat plants of both spring and winter cultivars were obtained from immature embryos bombarded after culture for more than 5 d, but not from those receiving less than 5 d. The integration and inheritance of the bar gene as a selectable marker gene were confirmed by the combination of PCR amplification and Southern analysis of T 0 and T 1 plants. Some of the bialaphos resistant plants showed the expression of the GUS gene co-transformed with the bar gene.

Altered expression of barley proline transporter causes different growth responses in Arabidopsis

A compatible solute, proline is accumulated in various kinds of plants and microorganisms under environmental stresses. The function of proline is thought to be an osmotic regulator under water stress, and its transport into cells is mediated by a proline transporter. Here, we report the effects of expressing the barley proline transporter (HvProT) under the control of either the CaMV35S promoter (35Sp) or a root cap promoter (RCp), on Arabidopsis growth. In Arabidopsis, transformed HvProT functions in the plasma membrane, like other amino acid transporters. Reduction in biomass production was observed in aerial parts of 35Sp-HvProT plants, and it was accompanied with decreased proline accumulation in leaves. Impaired growth of 35Sp-HvProT plants was restored by exogenously adding l-proline. These results suggested that growth reduction was caused by a deficiency of endogenous proline. In 35Sp-HvProT plants, the amount of proline dehydrogenase (PDH) transcript was increased compared to wild type (WT) plants, with a consequent enhancement of the activity of PDH. On the other hand, the transgenic RCp-HvProT plants accumulated 2- to 3-fold more proline in the root tip region compared to WT, and root elongation was enhanced at the same time. Thus, different physiological responses were caused by the altered location in accumulation of proline using two different promoters for heterologous expression of HvProT. These results indicate the importance of proline distribution at the tissue level during vegetative development.

Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants

Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear α(1,4)D-glucan polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic pathway.

Effect of six promoter-intron combinations on transient reporter gene expression in einkorn, emmer and common wheat cells by particle bombardment

Abstract The effect of six promoter-intron combinations on transient expression of β-glucuronidase (GUS) as the reporter gene was estimated in cultured einkorn ( Triticum monococcum ), emmer ( Triticum durum ) and common ( Triticum aestivum ) wheat cell lines. Four promoters; cauliflower mosaic virus (CaMV) 35S, tandem CaMV35S, maize alcohol dehydrogenase gene ( Adh1 ) and rice actin gene ( Act1 ) promoters, and two introns; Adh1 intron1 and castor bean catalase intron, were tested. Six vectors having different promoter-intron combinations were introduced into wheat cells by using particle bombardment. Different levels of GUS gene activity in three wheat calli were detected by an in situ enzyme assay. The CaMV35S promoter gave the lowest level of transient expression in wheat cells. On the other hand, the rice Act1 promoter showed the highest level of transient expression in all three wheat cells and was also the most efficient promoter of all, suggesting that the rice Act1 promoter is efficient for use in wheat transformation.

Fertile transgenic plants of Ipomoea trichocarpa Ell. induced by different strains of Agrobacterium rhizogenes

Cotyledon explants of Ipomoea trichocarpa Ell. were inoculated with ten strains of Agrobacterium rhizogenes. Hairy roots were produced from the cut surface of explants by inoculation with all bacterial strains. No clear differences in rhizogenicity were observed among the bacterial strains of A. rhizogenes tested. Whole plants were regenerated from the hairy roots transformed by all of the bacterial strains. These hairy root-derived plants exhibited the expected transformed phenotype, which was sexually transmitted to the progenies in Mendelian fashion as a single dominant locus. Transgenic I. trichocarpa plants possessing both the npt II and gus genes were also obtained from the hairy roots by infection with Agrobacterium rhizogenes containing the binary vector pBI121 in addition to the wild-type Ri-plasmid.

Chromosomal regions controlling anther culturability in rice (Oryza sativa L.)

Diallel analysis has revealed that anther culturability in rice (Oryza sativa L.) is a quantitative trait controlled by the nuclear genome. Mapping of anther culturability is important to increase the efficiency for green plant regeneration from microspores. In the previous study, we detected distorted segregation of RFLP markers in rice populations derived from the anther culture of an F1 hybrid between a japonica cultivar ‘Nipponbare’ and an indica cultivar ‘Milyang 23’. To clarify the association between chromosomal regions showing distorted segregation and anther culturability, the anther culturability of doubled haploid lines derived from the same cross combination was examined, and the association between alleles of the RFLP markers, which exhibiting the most distorted segregation on chromosomes 1, 3, 7, 10 and 11, and the anther culturability was evaluated. One region on chromosome 1 was found to control callus formation from microspores, and one region on chromosome 10 appeared to control the ratio of green to albino regenerated plants. In both regions, the Nipponbare allele had positive effects. Three regions on chromosomes 3, 7 and 11, however, showed no significant effect on anther culturability.